Myelin-reactive T cells are responsible for initiating the cascade of autoreactive immune responses leading to the development of multiple sclerosis. For better insights into the disease mechanism, it is of major importance to have knowledge on the sites at which these cells are active during disease progression. Hereto, we investigated whether it is possible to track myelin reactive T cells, upon labelling with SPIO particles, in the CNS of EAE animals by MRI. First, we determined the optimal labelling condition leading to a high particle uptake and minimal SPIO-PLL aggregate formation using Prussian blue stainings and inductively coupled plasma spectroscopy measurements. Results from labelling of rat derived myelin reactive T cells with low concentrations of SPIO particles (i.e. 25 μg/ml) in combination with different concentrations of PLL (0 – 1.5 μg/ml) showed that increasing amounts of PLL led to augmented levels of free remnant SPIO-PLL aggregates. In contrast, a low PLL concentration (i.e. 0.5 μg/ml) combined with high concentrations of SPIO (i.e. 400 μg Fe/ml) led to a high labelling efficiency with minimal amounts of aggregates. Second, the labelled myelin-reactive T cells were transferred to control rats to induce EAE. At the occurrence of hindlimb paralysis, the SPIO labelled myelin reactive T cells were detected in the sacral part of the spinal cord and shown to be highly confined to this region. However, upon transfer in already primed rats, transferred T cells were more widely distributed in the CNS and shown present in the spinal cord as well as in the brain. Our study demonstrates the feasibility to track SPIO labelled myelin-reactive T cells in the spinal cord as well as the brain of EAE rats upon systemic administration. Furthermore, we provide data on the optimal labelling conditions for T cells leading to a high particle uptake and minimal aggregate formation.